Improved transmission electron microscopy (TEM) of cultured cells through a "floating sheet" method

Abstract

Cultured cells provide isolated systems for both biochemical and morphological studies. Previous methods of processing cell culture specimens for electron microscopy (EM) have been limited to sectioning either a monolayer or centrifuged cell suspensions which are not morphologically intact. In our improved method, N-butylglycidyl ether is added to cell cultures (2-5 min with agitation) following in situ fixation (3.0% glutaraldehyde in 0.1 M Pipes, pH 7.2, for 20 min, osmium tetraoxide 4% for 20 min). A thin pliable "sheet" of cells floats free from the plastic culture device and can be manipulated (centrifuged or folded) to obtain a vast number of morphologically intact cells for examination. We have examined several cell types (vascular smooth muscle, lung, liver, and endothelial cells) grown in two types of plastic culture flasks (Nunc and Falcon). This new method provides excellent EM morphology, maximizes the number of cells examined, and allows determination of cell orientation since a remnant of the dissolved flask remains loosely bound to the bottom of the cells.